ABSTRACT More than half a million patients undergo surgeries to repair of large bony defects every year. However, the two primary options currently available for bone reconstruction are the use of bone harvested from the patient suffering from the injury by additional surgeries, or bone grafts from cadaver donors. However, these multiple operations incur significant morbidity on the patient. Thus, bone engineering offers an attractive alternative approach for regenerative medicine. Mesenchymal stem cells (MSCs) have a great potential for engineering bone grafts and have been shown to generate bone cells in-vitro in three dimensional (3D) scaffolds. However, the therapeutic application of human MSCs in bone regeneration and reconstruction is still limited by a physiological aging related decline in their differentiation potential, compromising the feasibility and reproducibility of therapies. Moreover, bone grafts generated from MSCs are only millimeters in size limiting their use in a clinical setting. Telomere shortening restricts cell proliferation and ultimately leads to in-vitro aging and loss of function. We have designed a highly innovative approach, to overcome these challenges based on preliminary data from our laboratory showing that the differentiation potency of aged MSCs can be restored by the factor ZSCAN4. Our overall goal is to design novel protocols generate bone grafts suitable for the treatment of degenerative bone diseases and bone reconstructive surgeries. In this application we will a. Establish a clinically scaled bone graft from tonsil derived human MSCs. b. Define the effects of ZSCAN4 on bone differentiation potential of aged and in vitro aged MSCs as a mean to allow the scalability to human bone size requirements. c. Determine the role of ZSCAN4 in regulation of stemness potency.